The broad, long term goal of the proposed research is to understand the structure and function of MHC proteins and of the accessory factors that help to load them with antigens. The first specific aim of the proposed research is to determine the three-dimensional structure of ER- associated aminopeptidase 1 (ERAP1) and the structural basis for its unusual length- dependent proteolytic activity. ERAP1 is a recently identified component of the class I antigen presentation pathway that is responsible for the final trimming of some peptide antigens so that they can be accommodated into the class I MHC binding site. How ERAP's enzymatic activity is regulated so that it trims longer peptides but preserves peptides of 8-9 amino acid residues is not known. In work directed at this aim, X-ray crystallography of ERAP alone and in complex with various inhibitors, allosteric activators, and accessory proteins will be used to help elucidate the structural and mechanistic basis for ERAP action. The second specific aim of the proposed research is to determine the immunological importance of peptide hairpins presented by class II MHC proteins, such as the one observed for an immunodominant HIV- gag peptide bound to HLA-DR1. The sequence determinants of hairpin formation, the structural basis for TCR recognition of the hairpin region, and the frequency of T cells recognizing hairpin structures will be determined through structural studies of MHC-peptide and MHC-peptide-TCR complexes, and MHC tetramer-mediated analysis of responding T cell frequencies. The third specific aim of the proposed research is to determine the mechanism of action of the peptide exchange and HLA-DM-modulation factor HLA-DO, through biochemical and biophysical approaches. Public Health Relevance Statement: These studies are relevant to public health because they will lead to better understanding of how the immune system becomes activated in health and disease. Detailed understanding of the molecular events that underlie antigen processing and presentation, and T cell recognition of MHC-peptide complexes, will help guide development of therapeutics and diagnostics intended to facilitate immune recognition of infectious agents and to discourage autoimmune activation and development of autoimmune diseases.